Study On Construction Of A Nanovaccine Against Spring Viremia Of Carp Virus And Its Adaptive Immune Mechanism

Spring viremia of carp(SVC)caused by Spring viraemia of carp virus(SVCV)is a highly infectious and lethal aquatic viral disease.Vaccination is an important means to prevent and control aquatic viral diseases,however,the immune effect and immune route are still the two core bottleneck problems of SVCV vaccine and other aquatic vaccines.Dominant epitope screening and vaccine delivery technology are important ways to solve the above bottleneck problems.In this study,immunoinformatics analysis and phage peptide library were used to screen effective antigen for SVCV.The SVCV nanovaccine(LSG-TDH)is consisted of filtered antigen,low molecular weight protein(LMWP),and tetralysine modified H-chain apoferritin(TDH).The biosafety of LSG-TDH was verified at the cellular and individual levels.After immersion vaccination,the immune effect of zebrafish against SVCV was evaluated.Moreover,the transmembrane transport mechanism of LSG-TDH nanovaccine and vaccine-induced adaptive immune mechanism were elucidated.The results obtained are as follows:1.Screening of SVCV dominant epitopeThe potential T lymphocyte epitopes and B lymphocyte epitopes of SVCV Glycoprotein(G)was predicted by immunoinformatics approach.The antigenicity,allergenicity and toxicity of predicted epitopes were further analyzed.Two T lymphocyte epitopes and 6 B lymphocyte epitopes were selected.On this basis,SVCV multi-epitope vaccine(MTPG)was designed and prepared by connecitig these selected epitopes.Meanwhile,the potential dominant epitopes of SVCV were also screened by phage library.After 4 rounds of panning,6 peptides(SV-1,SV-2,SV-3,SV-4,SV-5 and SV-6)with the high affinity were selected for solid phase synthesis.Zebrafish were vaccinated with these peptide vaccines via intraperitoneal injection,respectively.Serum antibody analysis and SVCV challenge test showed that SV-2 is the dominant SVCV epitope among these above peptides.Then we take MTPG,SV-2,and G131c previously screened by truncated method into account.MTPG,SMTPG(SV-2 in tandem with MTPG),and SG(SV-2 in tandem with G131c)were prepared by prokaryotic expression technology.Zebrafish were vaccinated with thses vaccine via intraperitoneal injection,respectively.Serum antibody analysis and SVCV challenge test indicated that SG showed significant immune protection effect,its immunoprotection rate reached 58%.2.Study on the construction and immune effect of SVCV nanovaccineFlow cytometry and in vivo imaging were used to evaluate the cell penetrate ability of widely-used cell penetrating peptides loaded with expgenous protein at the cellular and individual levels.LMWP could efficiently deliver expgenous protein into cells and zebrafish.On this basis,LMWP,SG and tetrpolylysine modified zebrafish H chain apoferritin(TDH)was connected by peptide linkers,and the recombinant protein(LSG-TDH)was expressed by prokaryotic expression system.The developed LSG-TDH could be self-assembled into nanocage with the size of ca.20 nm.The biosafety of the constructed vaccine was evaluated by cell viability detection,survival analysis,growth performance detection and tissue section observation.The results showed that the viability of EPC cells,HEK 293T cells,macrophages and ZF-4 cells were above 90%when the concentration of LSG-TDH vaccine was lower than 40 μg/mL,and there was no significant effect on the survival rate and growth performance between control and vaccinated zebrafish.After immersion vaccination,the vaccine-induced immune responses were evaluated using enzyme linked immunosorbent assay,real time-quantitative polymerase chain reaction,and viral challenge.The results showed that:LSG-TDH could significantly improve the activities of complement C3,superoxide dismutase,acid phosphatase and alkaline phosphatase in the mucus and serum of zebrafish,enhance the expression level of immune-related genes in the gill and spleen,promote the activation of antigen presenting cells in the gill and spleen,and induce the expression of specific antibodies IgZ and IgM in the mucus,gill,serum,and spleen of zebrafish.The immunoprotection rate of LSG-TDH reached 89%.3.Study on the delivery and transmembrane transport mechanism of SVCV nanovaccineThe delivery kinetics of LSG-TDH,SG and LSG in vitro(EPC cells and macrophages)and in vivo was studied by cell fluorescence,flow cytometry and in vivo imaging.The results showed that LSG-TDH possess significant mucous penetration and transmembrane transport ability.LSG-TDH could deliver into the spleen and kidney of zebrafish from the mucosal tissues such as gill,intestine,and skin.The content of LSG-TDH in the mucosal tissues(gill and intestine)and systemic tissues(spleen and kidney)was significantly higher than that in SG and LSG groups.The transmembrane transport mechanism of LSG-TDH on EPC cells and macrophages was analyzed by cell fluorescence,flow cytometry and chemical inhibitor experiments.The results showed that the entry of LSG-TDH into cells was time-dependent and energy-dependent.Chlorpromazine significantly inhibited the transmembrane transport of LSG-TDH.The contents of LSG-TDH into EPC cells and macrophages after treated with chlorpromazine decreased by 80%and 71%,respectively.The results indicated that the transmembrane transport of LSG-TDH was mainly achieved through clathrin-mediated endocytosis pathway.4.Study on the mechanism of adaptive immune response induced by SVCV nanovaccineTranscriptome sequencing technology was used to analyze gene expression in immune tissues and organs of zebrafish on days 28 after vaccination.The results showed that differentially expressed genes in gill and spleen were enriched in signal transduction,immune response,antigen presentation and receptor-mediated signal transduction.Combined with previous studies,we focused on exploring the adaptive immune mechanism mediated by TLR2.Molecular docking was used to analyze the interaction between SG and zebrafish TLR2.Immunocoprecipitation and yeast two-hybrid confirmed significant interaction between SG and zebrafish TLR2.By immunizing wild type zebrafish(WT)and TLR2 knockout zebrafish(TLR2-KO),in the gill and spleen lymphocytes of TLR2-KO,the protein expression level of various components of NF-κB and MAPK pathway devreased significantly.Moreover,the phosphorylation level of various components of mTORCl signaling pathway were significantly inhibited in the gill and spleen lymphocytes of TLR2-KO.Especially,the proliferation of specific B cells in the gill and spleen of TLR2-KO,the expression levels of specific antibodies IgZ and IgM in the mucus,gill,serum and spleen of TLR2-KO,and the immunoprotective effect of the vaccine in TLR2-KO were significantly reduced.These results suggest that TLR2 mediate LSG-TDH-induced adapyive immune responses by activation of antigen presenting cells.In conclusion,the constructed SVCV nanovaccine induce robust the adaptive immunity of zebrafish against SVC through the activation of antigen-presenting cells mediated by TLR2.This study not only provides a new strategy and reference for the construction of aquatic nanovaccine,but also provides a new perspective and new evidence for understanding the adaptive immune mechanism of teleost fish,which has important scientific significance and application prospect.